Glaucoma damage to retinal nerve fiber layer (RNFL) usually precedes detectable visual loss. Optical methods to directly assess the RNFL provide a promising way to detect early glaucomatous damage and have been used in clinical diagnosis of the disease. Although the optical properties of the RNFL in normal retina are well understood, glaucoma is likely to change axonal structures and little is known about the optical properties of RNFL under glaucoma development and progression. Our long-term goals are 1) to provide comprehensive and quantitative understanding of changes of the optical properties of RNFL in the context of glaucoma, 2) to study the anatomic origins underlying these changes, and 3) to translate this knowledge into improvements in measurement sensitivity for glaucomatous damage. An animal model of glaucoma will be used to study the reflectance and birefringence of RNFL and the distribution of axonal cytoskeletal components across the retina at different stages of glaucoma. The relation between changes in the optical properties and subcellular structure will also be investigated. RNFL reflectance arises from light scattering by cylindrical structures in axons. At least two mechanisms, modeled as arrays of thin and thick cylinders, contribute to the reflectance spectrum. Because glaucomatous damage will change the number and arrangement of axonal structures, it is expected to have wavelength dependent effects. Specific Aim 1 is to measure the RNFL reflectance spectrum for different stages of glaucoma. The results will reveal the wavelengths that are most sensitive for detecting RNFL defects and may provide insight into damage mechanisms. Microtubules are the major cytoskeletal component contributing to RNFL birefringence. Because subcellular change may precede irreversible loss of axons, change of RNFL birefringence may provide an early indicator of glaucomatous damage. Specific Aim 2 is to determine the sensitivity of birefringence change for detecting axonal defects in glaucoma by measuring the distribution of RNFL birefringence changes in the context of glaucoma. Glaucoma causes interruption of axonal transport. Cytoskeletal F-actin and MTs are responsible for various cellular processes including intracellular transport. Glaucoma, therefore, is expected to change F- actin and MT distributions. Because F-actin affects the structural organization of axonal MTs, Specific Aim 3 is to test the hypothesis that F-actin distortion precedes MT loss. The distributions of F-actin and MTs across the retina will be studied at different stages of glaucoma by simultaneous staining of both cytoskeletal components. The knowledge gained in this research will increase understanding of the mechanisms of glaucomatous damage and, ultimately, will improve assessment sensitivity for RNFL damage in clinical practice. PUBLIC HEALTH RELEVANCE: Glaucoma, a leading cause of blindness in the US, damages the retinal nerve fiber layer (RNFL). This project will provide comprehensive and quantitative understanding of the optical properties and underlying mechanisms of RNFL damage during the development of glaucoma. The ultimate goal of this project is to improve assessment sensitivity for RNFL damage in clinical practice.